Genomic epidemiology and multilevel genome typing of Bordetella pertussis

ABSTRACT Bordetella pertussis causes pertussis (or whooping cough), a severe respiratory infectious disease in infants, although it can be prevented by whole cell and acellular vaccines. The recent pertussis resurgence in industrialised countries is partly attributed to pathogen adaptation to vaccines, while emergence of antimicrobial resistance, specifically to macrolides in China, has become a concern. Surveillance of current circulating and emerging strains is therefore vital to understand the risks they pose to public health. Although the use of genomics-based typing is increasing a genomic nomenclature for this pathogen has not been well established. Here, we implemented the multilevel genome typing (MGT) system for B. pertussis with five levels of resolution, which provide targeted typing of relevant lineages and discrimination of closely related strains at the finest scale. The lower resolution levels (MGT2 and MGT3) describe the distribution of major vaccine antigen alleles including ptxP, fim3, fhaB and prn, as well as temporal and spatial trends within the B. pertussis global population. Mid-resolution levels (MGT3 and MGT4) enable typing of antibiotic-resistant lineages and Prn deficient lineages within the ptxP3 clade. The high-resolution level (MGT5) can capture finer-scale epidemiology such as outbreaks and local transmission events, with comparable resolution to existing genomic methods of strain-relatedness assessment. The scheme offers stable MGT-type assignments aiding harmonisation of typing and communication between laboratories. The scheme is available at https://mgtdb.unsw.edu.au/pertussis, is regularly updated from global data repositories and accepts public submissions. The MGT scheme provides a comprehensive, robust, and scalable system for global surveillance of B. pertussis.


Introduction
Bordetella pertussis causes the respiratory infectious disease, pertussis (whooping cough) which is particularly severe in infants. Globally, 24 million cases and 160,700 deaths were estimated to have occurred in 2014 [1]. A whole cell vaccine (WCV) was introduced in the late 1940s to early 1950s which significantly reduced disease incidence and mortality [2,3]. Due to side effects of the WCV, an acellular vaccine (ACV) was subsequently introduced in the 1990s. Importantly, re-emergence of pertussis has been observed in the last two decades in countries with high vaccine uptake.
Studies have suggested that the selection pressure from vaccine-induced immunity has acted on B. pertussis evolution in recent years [4][5][6][7][8], and therefore the use of ACV may have been a significant contributor to the resurgence of pertussis [9]. Increased variation of ACV-antigen genes, a mismatch of alleles between the vaccine strain and the current circulating B. pertussis strains and the recent increase in Prn-negative isolates all point to vaccine selection [4][5][6][7][10][11][12].
B. pertussis may also be under selective pressure from macrolide antibiotics used for treatment of pertussis such as erythromycin or azithromycin [13]. Macrolide-resistant strains of B. pertussis have recently emerged from the genetic background of ptxP1 strains in China [14][15][16] and very recently from the ptxP3 background [17,18]. Although only a small number of macrolide-resistant B. pertussis isolates have been reported in other countries, monitoring of the international spread of macrolide-resistant strains is of paramount importance [16].
Molecular techniques previously used for public health surveillance of B. pertussis each had shortcomings. Pulsed-field gel electrophoresis (PFGE) is timeconsuming and can be difficult to compare between studies while multi-locus variable-number tandemrepeat analysis (MLVA) and traditional 7 gene multi-locus sequence typing (MLST) both lack resolution in B. pertussis [19,20]. Allele typing of vaccine antigen genes and surface protein genes offered improved resolution and is widely used [21][22][23][24].
However, all of these methods lack the resolution for fine-scale typing required for detailed epidemiology in B. pertussis due to its very low genetic diversity.
A genotyping scheme based on single nucleotide polymorphisms (SNPs) divided B. pertussis into 42 SNP profiles (SPs) and six SNP clusters [4]. Subsequently, six epidemic lineages (ELs) were defined to describe local epidemic population structures within Australian SNP Cluster I isolates [25][26][27]. Although these typing method provided a granular representation of the B. pertussis population, they required manual curation and construction of phylogenetic trees for the description of the epidemic lineages.
Recently core and whole genome MLST schemes (cgMLST and wgMLST) have been developed for B. pertussis [28][29][30]. These schemes provide high resolution typing, with the majority of isolates assigned a unique identifier. Additional analyses are then required to group these identifiers together to describe larger trends, which add complexity and require technical expertise. We previously developed a genome typing method called multilevel genome typing (MGT) and have applied it to several bacterial species of public health importance [31][32][33][34]. MGT consists of a series of MLST schemes (or levels) with increasing numbers of loci. Each isolate is assigned a sequence type (ST) at each level providing a simple nomenclature with a gradient of resolutions. In this study, we developed an MGT scheme and an online database for B. pertussis and demonstrated its usefulness in describing both known and novel population structures and epidemiological trends.

Genomes used in this study
The initial set of genomes used to create the MGT scheme was made up of 4797 genomes. Of these, 4610 genomes passed MGT locus calling thresholds and were used to develop the MGT scheme [33]. These included 733 complete assemblies and 3877 sets of paired-end Illumina reads collected from the NCBI RefSeq database and sequence read archive (SRA), respectively. A large number of isolates with pairedend Illumina reads were released after the MGT scheme was generated. These 1930 sets of paired-end Illumina reads were added to previous datasets for a total of 6540 isolates.

Core genome definition including partial locus selection
Partial locus selection involved the division of reference loci in order to increase the proportion of isolates in which a locus can be reliably typed (typeability) and was composed of two steps. Firstly, all coding regions and intergenic regions from the B. pertussis Tohama I reference genome (GCF_000195715.1) with IS removed were initially selected and alleles were called as described previously (Supplementray methods A) [33]. Secondly, loci with regions that were poorly typable were split on either side of that region (supplementary methods B). If the resulting sections of the locus (called subloci) were greater than 100 bp, they were included as two or more separate loci in the core genome.

MGT level locus selection
The low diversity and high clonality of B. pertussis results in many loci with only 2 alleles across the entire species. These loci are ideal for MGT definition because, when combined into one MGT level, they will not produce multiple minor STs caused by rare alleles in a small number of isolates. To best describe a clade in the overall population a locus should have as close to 2 alleles as possible, one for the clade of interest and another for the remainder of the population (supplementary Figure 1). These loci were termed "phylogenetically informative loci" and made up MGT levels 2, 3 and 4. Combining one locus for each target clade into one MGT level ensures that each clade should have a distinct ST with minimal small STs introduced by sporadic mutations. Additional MGT level four loci were selected if they had at least 2 alleles, and all alleles were assigned to a minimum of 5 genomes. Finally, for a locus to be included an allele must be callable in all but 10 genomes (of 4,610) and must be partially callable in all but 20 genomes. In total an allele must be called in 4580/4610 genomes for a locus to be included in the MGT scheme.

MGT assignment
MGT assignment used pipelines described previously and is hosted at the MGTdb [33,34] (supplementary methods C).

Phylogenetic analyses
The allele profiles generated for each isolate that was successfully assigned an ST in the cgMLST scheme (n = 4610) were used to produce a phylogeny of the whole dataset using GrapeTree v1.5.0, in which the rapidNJ algorithm was used [35]. A phylogeny composed of a single representative for each of the 82 MGT4 STs assigned to more than 5 genomes was used to simplify visualisation of MGT levels and other typing systems. The phylogeny was generated by extracting SNPs from each MGT5 allele relative to the reference allele (allele 1) for each isolate. SNPs were then concatenated into an alignment that was used to generate a maximum likelihood phylogeny using IQ-TREE v2.2.0 with default settings, 1000 bootstrap pseudoreplicates and the K3Pu+F+I model automatically selected as the best-fit [36].

prn gene mutation detection
Disruptions of the prn gene including SNPs, insertions, deletions and IS disruptions were all examined using a combination of methods and were compared to previously described events (supplementary methods F).

B. pertussis core genome definition
Using 4610 genomes, 4633 core loci were identified with a total nucleotide length of 3.  Table  1). This core genome is much larger than the existing cgMLST scheme (1.75 Mbp) and comparable to the existing wgMLST scheme (3.34 Mbp) [28,30].
To facilitate locus selection for the MGT scheme levels, alleles were called for all core loci in the initial set of 4797 isolates using standard MGT scripts. A minimum threshold of 96% of core loci must be successfully assigned an allele in order for an ST to be assigned to a genome [33]. This threshold was reached in 4,610 isolates and these were used in the further development of the MGT scheme. In total 9875 new alleles were assigned to these isolates including 8408 (85.1%) in genes and gene fragments and 1467 (14.9%) in intergenic regions and intergenic region fragments. The average number of missing loci per genome is also an important measure of the typeability of the core genome. The core loci defined here had an average of 12.21 loci missing per isolate for 4797 isolates. The existing cgMLST scheme had an average of 14.4 loci missing per isolate for 1949 isolates [28]. Thus, the core loci allele typing performed here was reliable whilst providing significantly better resolution (Supplementary materials A).

B. pertussis MGT scheme design
Due to the low genomic diversity observed in current B. pertussis isolates, five MGT levels were implemented rather than the eight or nine levels defined in other species [31][32][33]. The first level is the well-established seven gene MLST [37]. MGT2, MGT3 and MGT4 loci were selected to best describe previously defined genotypes, clusters and lineages [4,[25][26][27] (Figure 1). MGT4 loci were also partly selected to minimise small STs (assigned to five or fewer isolates). MGT5 (cgMLST) loci were identical to the entire set of core genome loci defined above. Characteristics of each level are presented in Table 1.

MGT STs describe previously established typing schemes
To allow comparison to existing studies the degree of agreement between existing typing systems and MGT STs was compared. MGT STs from MGT2 and MGT3 were able to describe alleles in the ACV antigen genes (ptxA, fim2, fim3, prn, fhaB) and the pertussis toxin promoter (ptxP) with 100% specificity and over 90% sensitivity (supplementary results B and C and supplementary materials Table 3). Three SNP clusters and the majority of SPs defined previously [4] were well described by MGT2 and MGT3 STs (Figure 1(D and E)), while for the 6 Australian ELs [25][26][27], MGT4 STs provided highly specific typing (supplementary results D).

B. pertussis global population structure described using the multiple levels of MGT
An updated global dataset of 6540 isolates (including the MGT definition dataset of 4610) was used to examine the genetic and geographical distributions of B. pertussis populations using MGT (Supplementary Table 3). The most common MGT2 ST was ST2 (ptxP3 allele), making up 82.3% (5382/6540) of the entire dataset followed by ST7 and ST6 with 9.8% (644/6540) and 3.2% (207/6540), respectively. Overall 9 MGT2 STs containing more than 10 isolates accounted for 99.1% (6480/6540) of the dataset.
When applying the increased resolution of MGT3, 24 STs contained more than 10 isolates each and described 95.9% of the total sampled population (6274/6540, coloured nodes in Figure 2(A)). Importantly for the usefulness of these subdivisions, the large MGT2 ST2 was separated into 13 MGT3 STs, with the largest of these (ST7) making up 23.9% (1565/6540) of the total dataset. Interestingly, 98.0% (1166/1190) of all MGT3 ST12 genomes were found in North America while 98.5% (469/476) of MGT3 ST30 isolates were from China.
By further subdividing the population with MGT4 STs, more detailed country associations were identified ( Figure 2(B)). Many MGT4 STs contained isolates from multiple continents, such as ST8, ST9 and ST86. However, a number of STs were more strongly associated with single countries (over 90% of samples from the same country). For example, in the USA, this included multiple MGT3 ST12 subtypes as mentioned above, but also three MGT3 ST8 subtypes, MGT4 ST12, ST14 and ST16. Other country associated STs included three in Japan (MGT4 ST183, ST146 and ST114), three in China (MGT4 ST90, ST95 and ST96) and four in Australia (MGT4 ST98, ST10, ST113 and ST106). Although France had the second-highest number of sampled isolates (1041), there were no MGT4 STs uniquely associated with that country.

Exploring population structure changes within the ptxP3 clade over time using MGT
In addition to establishing spatial population differences, we also examined how the levels of MGT can describe temporal changes in population structure using ptxP3 strains (MGT2 ST2) [5,10,38]. MGT2 ST2 increased globally over time, making up 24.8%    (Figure 3(C)).

Tracking macrolide-resistant strains in China and globally
Macrolide-resistant isolates were identified by typing the resistance conferring mutation, A2047G, in the 23S rRNA genes.  Table  4, supplementary results E). Importantly two isolates from Taiwan and one isolate from Japan belong to Chinese resistant lineages (MGT4 ST90 and MGT4 ST96 respectively). Importantly, two macrolide-resistant Chinese isolates were identified within MGT2 ST2 (ptxP3). These isolates were within MGT3 ST7 but had different MGT4 STs (ST60 and ST446). The two isolates also fall into distinct branches on a phylogeny, separated by numerous erythromycin-sensitive isolates (data not shown). These results suggest that these isolates represent two independent mutational events leading to macrolide resistance.

Multiple independent Prn deficient lineages described by MGT
The rapid increase of Prn deficient isolates (Prn-) in the last 15 years has been associated with multiple independent mutational events across the B. pertussis population [6,39]. Using known mutations that silence Prn production, we examined all 6540 isolates and identified 2963 isolates (45.3%) as Prn-. These isolates were found in 15 of 33 major MGT3 STs (2919 Prnisolates) and 59 of 88 major MGT4 STs (2857 Prn-isolates) (Figure 4). Prn deficiency in these isolates was caused by 24 previously described mutations, 21 of which were previously confirmed by the Western Blot method (Supplementary Tables 3 and 5, Supplementary Figure 4). Of these mutations, 13 were assigned to more than 10 isolates (2833 isolates total) and 11 were assigned to 10 or fewer isolates (34 isolates total). An additional 200 isolates contained mutations whose effect on Prn production was unknown (marked uncertain).
Prn-isolates are in the majority in many MGT3 and MGT4 STs. At MGT3 the two previously described USA dominated lineages (MGT3 ST12 and MGT3 ST8) were almost entirely Prn-, however these deficiencies were not caused by the same mutation. In MGT3 ST12, 99.92% (1190/1191) were Prn-with 99.66% (1187/1191) being caused by event 24 (an IS insertion at position 1608). Within MGT3 ST8 three large MGT4 STs were predominantly Prn-but these deficiencies were caused by separate mutation events, for MGT4 ST12 (100% Prn-) event 12 (a nonsense mutation at position 1273), for MGT4 ST14 (96.53% Prn-) event 24 and for MGT4 ST16 (90.20% Prn-) event 56 (an IS insertion and deletion at position 240). Interestingly, event 24 is found in 3 major MGT2 STs, 7 major MGT3 STs, 21 major MGT4 STs suggesting that it has independently occurred numerous times. MGT STs can therefore describe multiple lineages that were dominated by a single causative mutation as well as distinguish between Prn-lineages with different causative mutations.
The six previously defined Australian epidemic lineages [26,27] can be described using MGT4 STs ( Figure 5(B)). EL1, EL2, EL5 and EL6 were assigned to MGT4 ST88, ST10, ST106 and ST56 respectively. EL3 and EL4 were assigned to 3 and 2 MGT4 STs respectively, all of which were found within MGT3 ST7. The division of EL3 and EL4 into multiple STs was expected as these ELs contained more than one independent sublineage [25].
MGT4 allows the description of temporal patterns of the ELs as well as describing diversity within EL3 and EL4 (Figure 5(C)). EL5 and EL6 STs were not observed after 2011 while the remaining ELs were observed in most years. Within EL3, MGT4 ST59

MGT5 provides high resolution typing for local-scale epidemiology
MGT5 provides high resolution typing for discriminating closely related isolates, which is vital given the low diversity of the B. pertussis population. Of the 6540 isolates in the global dataset, 3580 (54.74%) were assigned to singleton MGT5 STs while the remaining 2960 isolates were assigned to 720 MGT5 STs, each containing 2-75 isolates. We therefore investigated whether these STs could be used to infer relationships on small temporal and spatial scales.
To determine whether isolates sharing an MGT5 ST were more closely distributed than other isolates, pairwise comparisons of Australian isolate locations and dates were performed (n = 268). Previous work identified that isolates within 1km of each other and isolated within 18 months had increased relative risk of transmission [25]. Of isolate pairs with the same MGT5 ST, 46.10% (284/616 pairs) were isolated within 1km of each other and within 18 months of each other. By contrast for isolate pairs with different MGT5 STs, this same parameter was 0.20% (144/ 71208 pairs). Therefore, isolates that were assigned to the same MGT5 ST were far more likely to represent recent, local transmission events than isolates assigned different MGT5 STs. This trend is also found when examining the frequencies of temporal and spatial distributions for all pairs of isolates within  Figure 5B).
The performance of MGT5 relative to a previous cgMLST scheme was evaluated using 55 isolates examined in that study [28]. Of the 55 isolates examined in the previous cgMLST scheme 6 STs were defined with more than 1 isolate (supplementary Table 8). Of these STs, MGT5 was able to provide additional differentiation in 3 STs. In two other cases MGT5 grouped 2 isolates together that were separate in the previous scheme.

B. pertussis MGT web database features
The B. pertussis MGT scheme is publicly available at https://mgtdb.unsw.edu.au/pertussis and the database includes all isolates in this study. The database is also open to public data submission including raw reads and processed allele files and all publicly available B. pertussis isolates are continuously updated as they are released from SRA (Supplementary materials G). For publicly available isolates and raw read submissions, MGT types, vaccine antigen alleles and macrolide resistance SNP detection are called and reported per isolate. For processed allele submission, only MGT types and vaccine antigen calls are performed. The MGT database has an array of data export and visualisation features [34] that can facilitate epidemiological surveillance and outbreak investigation.

Discussion
This study presents a novel MGT scheme for B. pertussis capable of providing analyses ranging from species-wide population structure (MGT2) to high-resolution genotyping enabling cluster/outbreak investigations within one country (MGT5). These MGT sequence types offer a simple nomenclature for communication and standardisation allowing integration and description of previously disparate datasets and analyses. Importantly, the existing, widely used vaccine antigen gene allele nomenclature as well as other SNP-based typing systems were generally consistent with the MGT, allowing backwards compatibility with non-genomic studies and surveillance.
The MGT scheme has several advantages over other systems for assigning genomic types to B. pertussis isolates. A genomic typing database using cgMLST already exists in Institut Pasteur [28]. However, this database only provides typing using 7 gene MLST (equivalent to MGT1), vaccine antigen typing and cgMLST. As demonstrated in this study, the addition of MGT2 to MGT4 bridges a gap in epidemiological descriptions between these existing typing methods. Furthermore, in contrast to the Institut Pasteur database, our database is updated automatically with newly released data, making it easy for users to examine the most recent results as well as compare them with their own data. Other methods for assigning genomic types include non-database-based programs such as RhierBAPS. RhierBAPS uses Bayesian analyses to identify hierarchical clades within a phylogenetic population structure. Theoretically, RhierBAPS could identify the same clades as described by MGT2, MGT3 and MGT4 however the makeup of these clades and the identifiers assigned to them would depend on the dataset the program was run on and would not provide a stable and standardised nomenclature for communication and comparison of epidemiological trends.
Given the low genomic diversity of the B. pertussis population, it is essential to utilise the largest possible proportion of the genome to provide maximum discrimination between closely related isolates. MGT provides this high resolution as it utilised intergenic regions as well as fragments of genes to increase the overall resolution of the system. Due to these attributes, the MGT5 level (B. pertussis cgMLST) has equivalent resolution to an existing wgMLST scheme while maintaining the standardisation that is inherent in a cgMLST scheme [28,30]. The performance of MGT5 relative to the existing cgMLST scheme demonstrates that this increased resolution allowed closely related isolates to be further differentiated. The cases where an MGT5 ST was split into 2 cgMLST STs were likely due to differences in allele calling algorithms that lead to mutations in one scheme not being called in another. The performance of MGT5 typing in describing isolates that are closely related temporally and spatially suggest that they will be useful in describing fine scale epidemiology.
As well as providing high resolution at MGT5 the lower levels of the MGT (MGT2, 3 and 4) have been designed to describe lineages of interest and to match the underlying genetic relationships of the population as closely as possible. The ability to generate definitive, stable sequence types at multiple resolutions provides an effective system for surveillance of strain movement nationally and internationally. This is especially the case for lineages that are not frequently observed in more than one location. For example, MGT3 ST30, and its three resistant lineages, MGT4 ST95, ST96 and ST90, were almost exclusively found in China [16]. Given that macrolides are the first line treatment for pertussis and are also used as prophylaxis [40], the spread of macrolide-resistant strains internationally could have significant global public health impacts. Effective surveillance for these lineages is therefore essential and MGT can perform this role without requiring reanalysis of the whole dataset as demonstrated by the description of small numbers of these lineages in Japan and Taiwan. Similarly, MGT3 ST12 is almost entirely restricted to the USA where it is the most prevalent type. While any differences in the virulence and severity of disease caused by this clade have not yet been established, its dominance in the USA and potential international spread would also be of interest.
Recent studies have also identified ptxP3 (MGT2 ST2) isolates in China that carry the macrolide resistance conferring mutation [17,18]. Two such isolates are present in the MGT database and were both located within the globally distributed MGT3 ST7 with different MGT4 types. These isolates are of particular concern given that ptxP3 isolates are known to have a fitness advantage over non-ptxP3 isolates [6,41]. The three macrolide-resistant lineages described above were of ptxP1 background, which may not have enough fitness advantage to spread to countries where ptxP3 strains replaced ptxP1 strains. However, macrolide-resistant isolates within ptxP3 would competently compete with ptxP3 strains already circulating in those countries, especially during periods of epidemics when macrolide usage may be temporarily increased, making the surveillance of macrolide-resistant ptxP3 strains especially important.
Significant sampling of the B. pertussis population with genome sequencing is limited to only a few countries resulting in the overall dataset being unavoidably biased. However, conclusions relating to temporal and geographical ST distributions can still be drawn using comparisons of these more robustly sampled locations. For example, the geographic restriction of MGT3 ST12 to the USA could be explained by lack of sampling in other locations. However, in this case, three other MGT3 STs (ST7, ST11 and ST28) emerged within the same 4-year period (2008-2011) were found in multiple well sampled countries such as Australia, UK and France, suggesting that MGT3 ST12 had not spread to these countries. The reason for this lack of spread is unclear. The same was likely to be true for the Chinese restricted MGT3 ST30. These observations suggest that there are populations that are geographically restricted and maintained over at least 10 years in some locations. By contrast, no large geographically restricted STs were observed in France despite similar numbers of isolates being sampled. The reasons behind these differences are currently unclear.
The effective surveillance of ptxP3 strains (MGT2 ST2) and their subtypes is vital given its global dominance. Existing nomenclatures, such as fim3 alleles or SNP based approaches, have been used to describe the evolution of different groups within ptxP3 [4,6,[25][26][27], however each has limitations. fim3 alleles can only divide ptxP3 into 2 types therefore providing only a marginal improvement in resolution. SNP based approaches work well, but they are static. New clades identified after the SNP-based approach's development may not be described at all. In addition, they are currently not incorporated into a typing tools or maintained in a database. Consequently, they often require manual analysis and type assignment.
By contrast MGT typing can divide the ptxP3 strains into numerous subtypes from MGT3 to MGT5, which enabled detailed description of their epidemiology. For example, Prn deficient isolates have been shown to have a fitness advantage [6,42] and have increased in frequency in both Australia and the USA. However, the genetic cause of the loss of prn production is not always the same between these countries indicating that these strains were of independent origin. By applying MGT, it becomes clear that the increase in Prn deficient strains in the USA was caused by STs that are mostly absent in France and the rest of the world despite one of the mechanisms of disruption (event 24) being shared.
We previously studied the molecular epidemiology of Australian B. pertussis using both gene typing and genome sequencing [25,27,38]. The strains responsible for the past 2 epidemics from 2008 to 2017 in Australia (grouped into epidemic lineages) can be clearly disentangled using STs at MGT3 and MGT4 levels. MGT allows for the integration of EL surveillance into a standardised global system while providing backwards compatibility for isolate assigned ELs. This ability to perform ongoing surveillance allowed the identification of 2008-2012 epidemic STs that are now likely extinct (e.g. MGT4 ST56 and ST106) and those that continued to cause infections in Australia (MGT4 ST88, ST10, ST113, and ST9). MGT also clearly demonstrates that the majority of isolates in STs after 2011 were Prn deficient, supporting previous work demonstrating the increased fitness of such strains [6,25,42].
In conclusion, this study developed an MGT system for B. pertussis and showed that different MGT levels provide a distinct and useful characterisation of the B. pertussis population. MGT level 2 can represent vaccine antigenic allele types, MGT level 3 can describe the evolution of the ptxP3 strains, while MGT level 4 can identify independent Prn deficient lineages, lineages that caused the recent Australian epidemic and the three distinct Chinese erythromycin-resistant lineages. MGT level 5 offers the highest resolution for examining small scale temporal and spatial epidemiology. The MGT typing platform is publicly available and contains a continually updated database that provides visualisation, data export and typing to facilitate the adoption of this technology.